U.S. patent application number 14/427049 was filed with the patent office on 2015-08-27 for apparatus and method for point-of-collection measurement of a biomolecular reaction.
This patent application is currently assigned to CORNELL UNIVERSITY. The applicant listed for this patent is CORNELL UNIVERSITY. Invention is credited to David Erickson, Seoho Lee, Matthew Mancuso.
Application Number | 20150244852 14/427049 |
Document ID | / |
Family ID | 50278618 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150244852 |
Kind Code |
A1 |
Erickson; David ; et
al. |
August 27, 2015 |
APPARATUS AND METHOD FOR POINT-OF-COLLECTION MEASUREMENT OF A
BIOMOLECULAR REACTION
Abstract
A system, methods, and apparatus for biomolecular measurements,
monitoring, and tracking uses a smartphone-based system. A
biological sample, including, but not limited to blood, saliva,
biopsy, or sweat, is collected on a modular diagnostic test
platform, which is then inserted into a smartphone accessory.
Optical, electrical, mechanical, or other means are used to
transduce a biomolecular binding event, including antibody,
aptamer, enzymatic, base-pair matching, or other biological
recognition reaction and communicate the results with the
smartphone. Some specific examples of targets include
25-hydroxyvitamin D, folic acid, DNA, or proteins from infectious
agents, and zinc. The result can then be presented quantitatively
or turned into a more consumer-friendly measurement (positive,
negative, above average, etc.), displayed to the user, stored for
later comparison, and communicated to a central hub location where
medical professionals can provide additional review. Additionally,
social media integration can allow for device results to be
broadcast to specific audiences, to compare healthy living with
friends, to compete in health based games, create mappings, and
other applications.
Inventors: |
Erickson; David; (Ithaca,
NY) ; Mancuso; Matthew; (Bohemia, NY) ; Lee;
Seoho; (Ithaca, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CORNELL UNIVERSITY |
Ithaca |
NY |
US |
|
|
Assignee: |
CORNELL UNIVERSITY
Ithaca
NY
|
Family ID: |
50278618 |
Appl. No.: |
14/427049 |
Filed: |
September 6, 2013 |
PCT Filed: |
September 6, 2013 |
PCT NO: |
PCT/US2013/058422 |
371 Date: |
March 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61699486 |
Sep 11, 2012 |
|
|
|
Current U.S.
Class: |
455/557 |
Current CPC
Class: |
H04M 1/72527 20130101;
G01N 21/78 20130101; G01N 33/48792 20130101; G01N 2035/00881
20130101; G01N 21/31 20130101; B01L 3/502715 20130101; G01N 2201/12
20130101; Y02A 90/10 20180101; G16H 40/63 20180101; G16H 10/40
20180101; G01N 2201/062 20130101; G16H 40/67 20180101; G01N
2015/0038 20130101 |
International
Class: |
H04M 1/725 20060101
H04M001/725; G01N 21/78 20060101 G01N021/78; G01N 21/31 20060101
G01N021/31 |
Claims
1. A method that enables a point-of-collection, quantitative target
measurement of a biomolecular reaction in a collected sample,
comprising: providing a modular, diagnostic test platform that
includes a sample collection region, a biomolecular reaction
region, and a control reaction region; collecting a sample at the
sample collection region; providing a smartphone and a smartphone
accessory, wherein the smartphone is programmed with a software
application that operatively couples the smartphone and the
smartphone accessory, further wherein the smartphone accessory is
one of operatively connectable to and operatively connected to the
smartphone, further wherein the smartphone accessory is adapted to
removeably retain the modular, diagnostic test platform including
the collected sample, further wherein the smartphone accessory
provides an ambient light-free environment for at least the
biomolecular reaction region and the control region of the
diagnostic test platform including the collected sample at least
during a reaction measurement phase of the method, further wherein
at least one of the smartphone and the smartphone accessory
includes a light source having an emission that can illuminate at
least the biomolecular reaction region and the control region of
the diagnostic test platform; instructing the software application
to activate the light source; making a ratiometric measurement of a
biomolecular reaction in the biomolecular reaction region and a
biomolecular reaction in the control reaction region; and
displaying on the smartphone a quantitative target measurement of
the biomolecular reaction in the collected sample.
2. The method of claim 1, wherein the biomolecular reaction region
and the control reaction region each include a plurality of
functionalized nanoparticles.
3. The method of claim 1, wherein the biomolecular reaction region
and the control reaction region are in the form of microfluidic
channels.
4. The method of claim 3, further comprising making the ratiometric
measurement of the biomolecular reaction in the biomolecular
reaction region and the biomolecular reaction in the control
reaction region in parallel.
5. The method of claim 1, wherein the biomolecular reaction region
and the control reaction region are in the form of serially
disposed sub-regions on the diagnostic test platform.
6. The method of claim 6, wherein the biomolecular reaction region
is disposed upstream of the control reaction region.
7. The method of claim 1, wherein the diagnostic test platform is a
single-use, disposable component.
8. The method of claim 1, wherein the smartphone accessory further
comprises a microcontroller operatively coupled to the software
application, the light source, and a photodetector, wherein the
light source emission is at a wavelength that corresponds to a
resonant absorbance peak of the pluralities of the functionalized
nanoparticles.
9. A smartphone system capable of performing a biomolecular assay
on an analyte, comprising: a smartphone component; a smartphone
application component; and a smartphone accessory component,
wherein the components are linked in a manner that enables a
quantitative biomolecular assay on an analyte disposed on the
diagnostic test platform component, further wherein the system is
configured in a manner to communicate a quantitative indicia of the
assay.
10. The smartphone system of claim 9, wherein the smartphone
application component is operatively linked to at least one of a
USB-type accessory, a Bluetooth platform, Wi-Fi, and a smartphone
input port.
11. The smartphone system of claim 10, wherein the smartphone
application component is in a mode such that the smartphone
component provides operational power for the smartphone accessory
component.
12. The smartphone system of claim 9, wherein the smartphone
application component has a user-operated trigger in an application
interface that, upon activation, is programmed in a manner to send
a signal to the smartphone accessory component requesting a readout
of an absorbance value of the analyte.
13. The smartphone system of claim 12, further comprising a data
memory structure, wherein the smartphone application component is
programmed in a manner to store the readout of the absorbance value
in the data memory structure.
14. The smartphone system of claim 11, wherein the smartphone
accessory component further comprises: a case; a microcontroller
that is powered by the smartphone component, disposed in the case;
a light source coupled to the microcontroller, disposed in the
case; and an optical sensor disposed in a spaced, opposing relation
to the light source, disposed in the case, further wherein the
light source is characterized by an emission wavelength
corresponding to the resonant absorbance peak of the
nanoparticles.
15. The smartphone system of claim 14, further comprising a
modular, diagnostic test platform that includes a sample collection
region, a biomolecular reaction region, and a control reaction
region.
16. The smartphone system of claim 15, wherein the modular,
diagnostic test platform includes a plurality of functionalized
nanoparticles characterized by a resonant absorbance peak.
17. The smartphone system of claim 16, wherein the light source
emission wavelength corresponds to the resonant absorbance
peak.
18. The smartphone system of claim 14, wherein the smartphone
accessory component further comprises an optical sensor-respective
pinhole aperture disposed adjacent the optical sensor and a light
source-respective pinhole aperture disposed adjacent the light
source, wherein the respective pinhole apertures are disposed in
adjacent, opposing relationship.
19. The smartphone system of claim 15, wherein the diagnostic test
platform component is removeably disposed in the smartphone
accessory component.
Description
RELATED APPLICATION DATA
[0001] The instant application claims priority to U.S. provisional
Application Ser. No. 61/699,486 filed on Sep. 11, 2012, the subject
matter of which is incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] Embodiments of the present invention relate generally to the
field of biomolecular diagnostics and, more particularly to
portable, point of collection diagnostic systems, methods, and
components for biomolecular reaction measurements, monitoring,
tracking, and reporting, including social media applications.
BACKGROUND
[0003] Modern efforts in medicine and healthy living involve the
delivery of personalized care and management to the patient. Due to
the high variance inherent in biology, including in diagnostic
criteria, treatment, and disease management, often the best
solution for one patient is far from ideal for another. Before
optimal treatment and healthy living for an individual can be
prescribed by medical providers, the first step is collecting
information about them; however, to date much of this data
collection relies on questionnaires and surveys, diagnostic tests
being prohibitively expensive, especially for groups that are not
an immediate risk. These sorts of human input are often highly
variable as they rely on a patient's ability to recall their past
behavior as well as their integrity and embarrassment in admitting
certain actions associated with (supposed) unhealthy living.
[0004] The cost and accessibility of traditional medical diagnostic
instruments can and needs to be improved. Currently, diagnosis of
disease can take days to weeks while results are sent off to a
laboratory, and many diseases still cannot accurately be detected.
Devices capable of quickly and accurately diagnosing multiple
conditions could be applied to situations ranging from nutrition
and vitamin management in first-world locales to antibiotic and
vaccine triage in third-world villages. If created and packaged
correctly, such devices could ease the burden on gateway
physicians, provide impoverished countries with now inaccessible
diagnostic capabilities, protect combatants from biological warfare
agents, and increase health care access to the average person.
[0005] One implementation of these state-of-the-art diagnostics is
as smartphone and/or tablet (i.e., portable computing) accessories
where the computational power, read-out, data storage, and
connectivity are provided by an existing device. The smartphone has
penetrated nearly all aspects of our lives, affecting how we
consume media including news and entertainment, how we track our
finances and pay for goods and services, and how we monitor our
health and fitness. However, for all of the benefits smartphones
have provided, there is still little or no direct connection
between smartphones and in vivo biochemistry. By enabling a direct
link between a smartphone and small molecule detection, monitoring,
and tracking, a number of new benefits could be realized in the
fields of medicine and healthy living, including, e.g., simple
diagnosis of disease and nutrient deficiencies; monitoring and
tracking of existing conditions; and social media-enabled healthy
living updates, competition, game playing, and mapping.
[0006] Suboptimal nutrition is one of the most acute problems
facing the developed and developing world today. Worldwide, there
are more disability-adjusted life years lost to malnutrition than
any other medical condition; it is reported that over 1,000,000
people die every year from vitamin A and zinc deficiencies, and 30%
of all cancers are related to poor diet (by comparison genetics and
obesity account for only 5% and 10% of all cancers respectively).
Optimal pre-natal maternal folic acid levels are well co-related
with a reduction in neural tube defects and evidence suggests that
fetal brain development is enhanced by docosahexaenoic acid (DHA)
intake. Micronutrient (i.e., vitamins and minerals) deficiencies
have been tied to dozens of different health conditions including
anemia, rickets, scurvy, cardiovascular disease, and cancer.
Additionally, recent work has linked vitamin deficiencies to
obesity, one of the major challenges facing the current
generation.
[0007] The Copenhagen Consensus has identified tackling vitamin and
micronutrient deficiencies as the most cost-effective intervention
to further global development and progress in published reports
since 2004. Domestically, the Institute of Medicine has concluded
half of older adults in the United States who had hip fractures had
serum levels of 25(OH)D less than 12 ng/mL; (25-hydroxyvitamin D
[25(OH)D] is considered to be the best indicator of vitamin D; and,
that levels below 20 ng/mL are inadequate for bone and overall
health. The vast majority of vitamin and micronutrient analysis is
done through blood collection via venipuncture, which is then sent
away to a centralized laboratory. This analysis is slow, expensive,
requires trained personnel, and is not widely available,
particularly in resource-limited settings where micronutrient
deficiency is most harmful. A combined HPLC-MS method is considered
the industry standard for vitamin D testing, however ELISA kits and
similar immunoassays are comparable in terms of sensitivity and
accuracy, while being better suited for adaption to home use. Since
micronutrient deficiencies are not often clinically obvious, these
tests are typically done at the insistence of the patient. The fact
that so many Americans are vitamin deficient testifies to the fact
that the current methodologies are not working.
[0008] Salivary cortisol is a routinely used biomarker of stress
and related psychological diseases. Commonly, cortisol is elevated
in patients who experience a sudden stressor and returns to normal
after a period of time whose length is dependent on the strength of
the stressor. In patients with chronic stress disorders, such as
PTSD, it has been difficult to co-relate absolute levels of
cortisol at any given time with the diagnosis of a disorder due to
the large number of confounders. A better approach would be to
track cortisol, and other biomarkers, over time to look for trends
that could be indicative of the onset psychological disease.
[0009] Every year hundreds of millions of people suffer from
infectious diseases including respiratory infections, HIV/AIDS,
diarrheal diseases, tuberculosis, and malaria. The agents that
cause these diseases, including bacteria, viruses, fungi, etc., are
often easily manageable with proper identification yet routinely go
undetected because of the costs and difficulties associated with
diagnostic technology. In some cases, such as tuberculosis,
identifying the disease rapidly and on location can allow for
preventative measures prohibiting the disease from spreading
further. In other cases, such as HIV, keeping an acute-eye on
antibody levels is critical in tracking the progress of the
disease.
[0010] Kaposi's sarcoma (KS) is an opportunistic infectious cancer
that first became widely known during the acquired immunodeficiency
syndrome (AIDS) epidemic of the 1980s. During this time period, the
appearance of symptoms of KS, red lesions on the skin, became signs
that an individual was infected with human immunodeficiency virus
(HIV) and KS itself became known as an AIDS-defining illness. As
the battle against AIDs waged on, the introduction of highly active
anti-retroviral therapy (HAART) helped reduce KS incidence. Years
later, however, HIV infected individuals still contract KS at a
higher occurrence than when compared to the pre-AIDS era. Today, KS
is the fourth leading cancer in sub-Saharan Africa, and in some
countries, such as Uganda, is the most prevalent cancer in men. The
root cause of KS is Human herpes virus 8 (HHV-8), more commonly
referred to as Kaposi's sarcoma associated herpes virus (KSHV).
While the virus is often asymptomatic in healthy individuals, a
number of populations, including those immune-compromised by HIV,
are vulnerable to its symptoms. The virus is commonly believed to
be transmitted through saliva and in some regions rapidly spreads,
beginning in childhood affecting large portions of the population,
reaching seroprevalence of over 50%. Like other herpes viruses,
KSHV can establish a latent infection and remains without causing
any disease for the remaining life in most infected hosts, being
necessary but not sufficient of KS development.
[0011] In the developed world, medical professionals diagnose KS
with sufficient accuracy. If typical hematoxylin and eosin
(H&E) staining are applied to a KS biopsy section a number of
unique features can be observed, including many and large vascular
spaces as well as high numbers of spindle cells thought to be of
lymphatic endothelial origin. However, due to the existence of
similarly presenting diseases, such as bacillary angiomatosis (BA),
identification of these features is not sufficient for diagnosis of
KS. In modern hospitals this is solved through immunohistochemistry
staining for protein markers of KSHV, or through application of PCR
for KSHV sequences. However, neither of these techniques is readily
adaptable for use in the developing world where KS is most
prevalent.
[0012] Finding a solution to the aforementioned types of challenges
and problems directly motivated the development of lab-on-a-chip
based point-of-care diagnostics, beginning some 15 years ago. The
technical vision behind these kinds of systems comprised two parts:
a consumable "chip" that contains microfluidics and a biosensor,
and a "reader" instrument that interprets the signal from the chip
and provides results to the operator. Since this vision was first
put forward, the technology has advanced at an incredible rate to
the point where we now have devices that can operate over a million
microfluidic valves in parallel, portable PCR machines for pathogen
detection, nanosensors that can detect a handful of molecules, and
numerous other systems. These developments have significantly
reduced the size of the sample required to perform a blood
analysis.
[0013] To date, little of this visionary technology has
transitioned to personalized nutritional and vitamin analysis, for
example. There are two reasons for this: first is the difficulty in
obtaining quantitative results with a simple one-off test. The
majority of commercially available point-of-use tests for the
consumer market are based on the lateral flow principle.
Unfortunately, these types of tests are only able to provide
non-quantitative information and are only useful when the desired
result is binary (e.g. pregnant/not-pregnant). Obtaining
quantitative results requires complex sensors and sample handling
techniques that typically must be interpreted and displayed by a
reusable reader. This leads to the second challenge: In the
marketplace, the reader/consumable model (e.g., the razor/razor
blades) has proven successful only where the user makes numerous
measurements over the course of a day or week (e.g., blood glucose
monitoring). When measurements are made sporadically or with much
lower frequency (as with vitamins) the cost of purchasing a reader
system is prohibitively high, even if the consumable can be made
relatively inexpensive.
[0014] The extreme societal penetration of smartphones holds the
potential to alter this predicament. It is predicted that by 2016
there will be 250 million smartphones in use in the US. A good
portion of the complexity required to make and interpret a
quantitative in-vitro measurement is already embedded in
smartphones, resulting in a paradigm shift in the "razor and
blades" model. Put simply, most consumers now already own the
expensive part, the "Razor," in the form of a smartphone; all one
needs then is the blades.
[0015] A number of systems have been developed. Examples include
fitness monitoring, vaccine logs, sleep monitoring apps, and skin
cancer diagnostics. Smartphones have been used to collect heart
rate, blood pressure, and blood oxygen saturation. In 2011,
smartphone-based healthcare was worth $1.3 billion, up nearly twice
its 2010 value. Importantly however, all these existing commercial
smartphone based systems rely on user input or physical
measurements that are generally non-specific to a particular
pathology.
[0016] The inventors have recognized that expansion beyond these
coarse measurements requires molecular analysis of bodily fluids
like sweat, saliva, urine and blood, all of which contain a much
deeper wealth of physiological information. The inventors have also
recognized a need for mobile, point-of-collection devices and
methods that address all of the challenges outlined above. Most
importantly, the inventors have recognized the benefits and
advantages of a smartphone-type system, components thereof,
associated methods, and applications for quantitative bimolecular
detection assays, embodiments of which will be described in detail
below. Regardless of the disease, nutritional deficiency, or other
foci of the measurement, rapid, point-of-collection,
smartphone-based detection could provide enormous benefits in terms
of the amount of information that could be provided to medical
personnel. The temporal resolution and time-charted measurements
provided by smartphone diagnostics could be critical in treating
individual patients and providing needed personalized care. The
geographic information and connectivity of smartphones could be key
in tracking diseases or other agents spreading through populations.
By leveraging the processing power, display, and other components
of a smartphone-type system, point-of-care diagnostics could be
made considerably less expensive than modalities currently
available.
SUMMARY
[0017] As used herein, the term `smartphone` or `smartphone-type
device/system` means a mobile apparatus that is capable of running
a programmed application suitable for executing the embodied
functionality. While suitable traditional smartphone or
smartphone-type devices may include, e.g., products such as the
iPhone (Apple, Inc.) or Android (Google Inc.)-based smartphones and
tablet computers, a smartphone or smartphone-type device (e.g.,
Apple iPad) as discussed and embodied herein need not include a
telephone, per se, and may or may not include a built-in light
source (e.g., flash) and/or camera/CCD and, therefore, may include
tablets or other devices having size and weight characteristics
similar to, or smaller/lighter than, conventional smartphones or
smartphone-type devices. Thus a `laptop` computer would not
necessarily be covered under the definitional use of the term
`smartphone;` nor would a computing device that could be made
`portable` or `mobile` by an accompanying apparatus that might give
it portability or mobility. For the sake of conciseness, the term
`smartphone` will be used herein (including the claims) to mean
smartphones or smartphone-type devices as discussed within the
paragraph above.
[0018] The term `rapid` as used herein (and in the claims) to
modify the phrase `point-of-collection, quantitative target
measurement of a biomolecular reaction in a collected sample` is
defined to mean `essentially in real time` (e.g., seconds,
minutes).
[0019] The term `point-of-collection` as used herein (and in the
claims) to modify the phrase `quantitative target measurement of a
biomolecular reaction in a collected sample` is defined to mean
making a rapid target measurement at the time a sample is collected
on a modular diagnostic test platform in possession of the user and
inserted into the possessed smartphone accessory, not at a later
time, for example, after a sample has been collected and sent to a
laboratory.
[0020] An embodiment of the invention is a method for measuring a
target of a biomolecular reaction in a biological sample using a
smartphone. Illustrative steps include: collecting a sample on a
diagnostic test platform; recognizing the sample; communicating
sample information (signal transduction) from the diagnostic test
platform to a smartphone sample analyzer component (smartphone
reader/accessory); and signal processing the sample for analysis.
Optional additional steps include interpretation and/or display of
the assay results on the smartphone and/or using a smart phone and
smart phone application for measurement, logging, tracking, and
sharing of measurement information enabled by smartphone
connectivity and social media. The embodied method may be
characterized by the following illustrative, exemplary,
non-limiting aspects:
sample collection through a finger prick to collect a droplet of
blood; sample collection through collection of saliva via ejection
or cheek swab; sample collection via urine, sweat, tear, or other
bodily fluid collection; sample collection through collection and
processing of a solid sample including a biopsy or DNA from a
biopsy; sample collection through collection and processing of gas
samples by collection of respiratory output; sample recognition
through antibody-antigen recognition; sample recognition through
DNA base pair recognition; sample recognition through
aptamer-target recognition; sample recognition through enzymatic
recognition; signal transduction through optical, electrical,
mechanical, or other means, including optical, electrical, or
mechanical signal amplification based on resonance or other means
taking place between a disposable test platform and a reader
accessory; signal transduction using surface enhanced Raman
spectroscopy to obtain a "fingerprint" of specific biomolecules;
signal transduction via an absorption measurement at specific
wavelengths, wherein biomolecular recognition is turned into a
change in these wavelengths via gold nanoparticle and/or other
nanoparticle-based assays; measurement, logging, tracking, and
sharing enabled by smartphone connectivity and social media;
detection via gold nanoparticles;
[0021] detection on a surface;
[0022] detection in a solution;
detection via comparing (ratiometric) an absorbance of a test assay
to that of a control;
[0023] providing a quantitative target measurement;
where the detected targets are vitamins, minerals, other
micronutrients, pH, hormones, viruses, bacteria, or factors
associated therewith (e.g., vitamin/nutrient deficiencies; stress
associated with cortisol levels; sodium concentration, others);
where the target detected is vitamin D, or any of its down-stream
metabolites, including 25-hydroxyvitamin D, 1,25-dihydroxyvitamin
D, or 25-hydroxycholecalciferol; where the targets detected are
pathogens, pathogen proteins, pathogen nucleic acids, or other
pathogen biomolecules;
[0024] where the biomolecules detected is KSHV DNA.
[0025] An embodiment of the invention is a smartphone-based system
capable of performing a biomolecular assay on an analyte. The
smartphone-based system includes a smartphone, a smartphone
application, and a smartphone accessory. According to various
non-limiting, exemplary aspects:
wherein the smartphone-based system further comprises a modular,
diagnostic test platform; [0026] wherein the smartphone accessory
includes a magnetic latch for removeably holding the modular
diagnostic test platform; [0027] wherein an LED light source is
disposed directly across from a photodiode optical sensor, both of
which are disposed behind respective pinhole apertures, in the
case; [0028] wherein the modular diagnostic test platform is a
disposable test strip; [0029] wherein the modular diagnostic test
platform is a reusable component; [0030] wherein the modular
diagnostic test platform has a collapsible microchannel
characterized by an aspect ratio wherein the microchannel is
collapsible into a nanochannel; [0031] wherein the modular,
diagnostic test platform includes a sample collection region, a
biomolecular reaction region, and a control reaction region; [0032]
wherein the modular diagnostic test platform includes a plurality
of functionalized nanoparticles; [0033] wherein the functionalized
nanoparticles are functionalized with single-ended strands of DNA;
[0034] wherein the functionalized nanoparticles are coated with an
oligonucleotide; [0035] wherein the functionalized nanoparticles
are functionalized with hydroxyvitamin D antibodies; [0036] wherein
the functionalized nanoparticles are functionalized with
anti-hydroxyvitamin D antibodies; [0037] wherein the modular
diagnostic test platform includes at least two groups of
functionalized gold nanoparticles which are each coated with a
different oligonucleotide; wherein the smartphone-based system is
capable of performing a quantitative biomolecular assay on an
analyte; the smartphone application is configured to allow the
smartphone to supply power for operation of the smartphone
accessory; the smartphone application is configured to provide
email functionality; the smartphone application is configured to
store results on an Internet or cloud-based file storage and
synchronization platform; [0038] storing the results as text files;
[0039] storing the results on a Google Drive account; the
smartphone application is configured to create keyhole mark-up
language files (KML files) that can be opened in Google Maps,
Google Earth, or other geographic applications; the smartphone
accessory (reader) is connectable to a smartphone, wherein the
smartphone accessory includes a case or housing; [0040] wherein the
smartphone accessory includes a smartphone connector; [0041]
wherein the smartphone connector is a .mu.USB connector; [0042]
wherein the smartphone accessory is connectable to a smartphone via
a Bluetooth platform, a USB-type accessory, Wi-Fi, and a smartphone
input port; [0043] wherein the smartphone accessory includes a
microcontroller; [0044] wherein the smartphone accessory includes
an optical sensor; [0045] wherein the smartphone accessory includes
a light source; [0046] wherein the light source is a LED; [0047]
wherein the light source is a laser; [0048] wherein the light
source has a wavelength corresponding to a resonant absorbance peak
of a gold nanoparticle (GNP); [0049] wherein the light source has a
wavelength centered around 520 nm; [0050] wherein the smartphone
accessory includes a spectrometer; [0051] wherein the smartphone
accessory includes a Raman detector.
[0052] An embodiment of the invention is a method that enables a
rapid, point-of-collection, quantitative target measurement of a
biomolecular reaction in a collected sample. The method includes
the steps of providing a modular, diagnostic test platform that
includes a sample collection region, a biomolecular reaction
region, and a control reaction region; collecting a sample at the
sample collection region; providing a smartphone and a smartphone
accessory, wherein the smartphone is programmed with a software
application that operatively couples the smartphone and the
smartphone accessory, further wherein the smartphone accessory is
one of operatively connectable to and operatively connected to the
smartphone, further wherein the smartphone accessory is adapted to
removeably retain the modular, diagnostic test platform including
the collected sample, further wherein the smartphone accessory
provides an ambient light-free environment for at least the
biomolecular reaction region and the control region of the
diagnostic test platform including the collected sample at least
during a reaction measurement phase of the method, further wherein
at least one of the smartphone and the smartphone accessory
includes a light source having an emission that can illuminate at
least the biomolecular reaction region and the control region of
the diagnostic test platform; instructing the software application
to activate the light source; making a ratiometric measurement of a
biomolecular reaction in the biomolecular reaction region and a
biomolecular reaction in the control reaction region; and
displaying on the smartphone or the smartphone accessory a
quantitative target measurement of the biomolecular reaction in the
collected sample. According to various non-limiting, exemplary
aspects, the method may include the following steps, features,
components and/or characteristics:
wherein the biomolecular reaction region and the control reaction
region each include a plurality of functionalized nanoparticles;
wherein the biomolecular reaction region and the control reaction
region are in the form of microfluidic channels;
[0053] further comprising making the ratiometric measurement of the
biomolecular reaction in the biomolecular reaction region and the
biomolecular reaction in the control reaction region in
parallel;
wherein the biomolecular reaction region and the control reaction
region are in the form of serially disposed sub-regions on the
diagnostic test platform;
[0054] wherein the biomolecular reaction region is disposed
upstream of the control reaction region;
wherein the diagnostic test platform is a single-use, disposable
component; wherein the smartphone accessory further comprises a
microcontroller operatively coupled to the software application,
the light source, and a photodetector, wherein the light source
emission is at a wavelength that corresponds to a resonant
absorbance peak of the pluralities of the functionalized
nanoparticles.
[0055] An embodiment of the invention is a diagnostic test
platform. The diagnostic test platform includes a sample collection
region, a biomolecular reaction region, and a control reaction
region. According to various non-limiting, exemplary aspects, the
diagnostic test platform may include the following, features,
components and/or characteristics:
the biomolecular reaction region and the control reaction region
each include a plurality of functionalized nanoparticles; the
biomolecular reaction region and the control reaction region are in
the form of microfluidic channels;
[0056] wherein the microfluidic channels are collapsible into
nanofluidic geometries;
wherein the biomolecular reaction region and the control reaction
region are in the form of serially disposed sub-regions on the
diagnostic test platform;
[0057] wherein the biomolecular reaction region is disposed
upstream of the control reaction region;
wherein the diagnostic test platform is a single-use, disposable
component.
[0058] Additional features and advantages of the invention will be
set forth in the detailed description to follow, and in part will
be readily apparent to those skilled in the art from that
description or recognized by practicing the invention as described
herein, including the detailed description which follows, the
claims, as well as the appended drawings.
[0059] It is to be understood that both the foregoing general
description and the following detailed description are merely
exemplary of the invention, and are intended to provide an overview
or framework for understanding the nature and character of the
invention as it is claimed. The accompanying drawings are included
to provide a further understanding of the invention, and are
incorporated in and constitute a part of this specification. The
drawings illustrate various embodiments of the invention and
together with the description serve to explain the principles and
operation of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] The present invention will be more fully understood and
appreciated by reading the following Detailed Description in
conjunction with the accompanying drawings, in which:
[0061] FIG. 1 is a high-level flow chart expressing the steps of a
method for measuring a target of a biomolecular reaction in a
biological sample using a smartphone, according to an embodiment of
the invention;
[0062] FIG. 2 is a photoreproduction showing a smartphone, a
smartphone accessory, and a modular diagnostic test platform with
an addition enlarged view of the diagnostic test platform,
according to an exemplary, non-limiting embodiment of the
invention;
[0063] FIG. 3 is a photoreproduction showing the smartphone
accessory and the modular diagnostic test platform illustrated in
FIG. 2 with the diagnostic test platform inserted in the smartphone
accessory;
[0064] FIG. 4 shows: a) a smartphone system as depicted in FIG. 2;
(b) a smartphone app user interface; (c) components of a smartphone
accessory as depicted in FIG. 2; and, (d) a disposable modular
diagnostic test platform, according to an exemplary , illustrative
embodiment of the invention;
[0065] FIGS. 5(a, b) show a smartphone accessory according to an
alternative exemplary aspect of the invention;
[0066] FIG. 6 schematically illustrates an alternative, exemplary,
modular diagnostic test platform; (a) shows a diagnostic test
strip, its components, and its operation; (b) illustrates the
possible outcomes of running a strip, which are imaged via the
smartphone system, according to an exemplary aspect of the
invention;
[0067] FIG. 7: a) schematically illustrates an alternative,
exemplary, modular diagnostic test platform (as illustrated in FIG.
6) with details of a competitive Vitamin D assay including blood
filtration; b) a more detailed view of stages two and three from
Fig. (a), according to an alternative exemplary aspect of the
invention;
[0068] FIG. 8 illustrates a mode of connection between a smartphone
and a smartphone accessory according to an illustrative aspect of
the invention;
[0069] FIG. 9: a) (left) Aggregated and (right) unaggregated gold
nanoparticle solutions are shown, as well as the absorbance of a
solution after the addition of varying amounts of target DNA; b)
The absorbance spectrums of five different concentrations of gold
nanoparticle conjugates; and c) The absorbance spectrums of (b)
used to compare readouts from the smartphone accessory to a
spectrometer, according to an illustrative aspect of the
invention.
DETAILED DESCRIPTION OF NON-LIMITING, EXEMPLARY EMBODIMENTS OF THE
INVENTION
[0070] An exemplary embodiment of the invention is a method for
rapidly obtaining and presenting (i.e., displaying or communicating
out) a quantitative target measurement of a biomolecular reaction
in a collected biological sample at the time (and location) of the
sample collection event using a specially adapted (embodied)
smartphone system. FIG. 1 shows, at a high level, the method steps
involved in making the aforementioned target measurement using a
smartphone system as depicted in FIGS. 2-4, and, alternatively,
FIG. 6, which method and apparatus will be described in greater
detail as follows.
[0071] An exemplary smartphone system 100 is shown in FIGS. 2 and 4
and includes a smart phone component 201, a smartphone accessory
component 202, and a smartphone software application 203
(illustrated by a user interface display). Also shown in FIGS. 2
and 3 is an illustrative embodiment of a modular diagnostic test
platform 204 designed to operate with the smartphone system, and
which forms a part of the embodied invention.
[0072] The smartphone component has been described herein above and
needs no further description to be well understood. The smartphone
accessory 202 connects to the smartphone 201 via USB (as
illustrated in FIG. 8), Bluetooth, Wi-Fi, or other supported
connection and acts as an interface between the computational
power, display, and connectivity of the smartphone along with the
biological interface of the modular diagnostic test platform 204.
As a bridge between the two components, the smartphone accessory
contains a microcontroller 407 (e.g., an 8 MHz Pro Micro Arduino;
FIG. 4) or equivalent component capable of communicating with the
smartphone and capable of controlling any number of actuators,
signal transducers, or other components. In an exemplary aspect
further illustrated in FIG. 4, the smartphone accessory contains an
LED 409 or other light source and a photodiode, photocell, or other
optical transducer 411, which with some additional optical
components that are not part of the invention per se functions as a
smartphone-based spectrometer. In alternative aspects, the
smartphone accessory could contain a laser, a spectrometer, and/or
a Raman detector.
[0073] As will be described in greater detail below, the light
source 409 has a wavelength that corresponds to the absorbance peak
of a functionalized nanoparticle reaction (in the examples to
follow, functionalized gold nanoparticles (GNPs) are used having a
resonant absorbance peak at 520 nm. The microcontroller 407
connects to a male .mu.USB connector in order to draw power from
and communicate with the smartphone. The smartphone accessory 202
is enclosed in a housing 415 to block out ambient light.
[0074] As further illustrated in FIG. 4, the housing 415 holds the
LED 409 directly across from the photodiode 411, both behind
pinhole apertures (not shown). When a modular diagnostic test
platform 204 (described in detail below) is inserted into the
accessory, the path between the two components is filled with a 1
cm path length fluidic channel full of a nanoparticle solution. An
optional magnetic latch 417 for aligning and securing the inserted
modular diagnostic test platform 204 is further illustrated.
[0075] In an illustrative, exemplary aspect, the microcontroller in
the smartphone accessory was programmed to communicate with the
smartphone when attached and then wait for a signal to collect
data. On receiving a signal, the LED was powered ON for five
seconds, and then a measurement of the voltage drop across the
photocell was made. The photocell was connected via a pulldown
resistor, allowing for measurements of its resistance by comparing
the drop in voltage across it to the drop in voltage across the
resistor. This resistance drop was then directly correlated to the
amount of light on the photocell, or as was done, the absorbance of
the solution (on the diagnostic test platform) between the light
source and photocell.
[0076] An alternative smartphone accessory 501 is illustrated in
FIGS. 5(a, b). In this illustrative aspect, the accessory was
created integral with a case that attached to the smartphone in
such a manner that the smartphone accessory 501 is disposed
functionally proximate to a built-in flash component of the
smartphone, which, with or without a diffuser/filter, functions as
the light source for detection of the target biomolecular reaction.
As schematically illustrated in FIG. 5c (1, 2, 3), The accessory is
opened, the modular diagnostic test platform including the
collected sample is inserted therein, and upon closure, a
measurement of the biomolecular reaction in the collected sample
made and displayed by the smartphone system.
[0077] In an illustrative prototype using an Android smartphone
platform, a software application 203 was developed in eclipse
(REF). The code was written in Java and used the Android SDK tools.
The software was written to communicate with a USB accessory in
host mode, allowing the Android device to provide power for the
smartphone accessory. When triggered via a button press in the
application interface (FIG. 4b), a signal is sent to the smartphone
accessory requesting a readout of the current absorbance. The
application then receives this read-out, displays it to the user,
and saves it in a temporary memory. As results are collected, they
can also be automatically tagged with user information, time
stamps, and locations to provide relevance to the results. In
addition to the application's core functionality, utilities were
added to email results to other experimenters or medical
professionals, to store results as text files on a connected Google
Drive account, and to create keyhole mark-up language files (KML
files) that could be opened in Google Maps, Google Earth, or many
other geographic applications. Using these functions together, a
simple database of test read-outs from numerous devices can be
created, expanding the applicability of the embodied invention.
[0078] An exemplary modular diagnostic test platform 203 (along
with the smartphone accessory in which it is designed to operate)
is illustrated in FIG. 3. The modular diagnostic test platform,
which in the illustrated aspect is disposable, acts as a sample
holder and contains all of the necessary components for
biomolecular recognition of one or more targets. A sample is
collected in a sample input region 301 (e.g., in the case of blood,
through a finger-prick similar to a glucose measurement).
Biological samples include, but are not limited to, liquids like
blood, tears, sweat, urine, or other bodily excretions, as well as
solid samples including biopsies. The modular diagnostic test
platform includes two microfluidic channels 304-1, 304-2 disposed
in parallel. Each contains functionalized nanoparticles and provide
structural and functional media (regions) for a biomolecular
reaction (304-1) and a control reaction (304-2). In FIG. 3, a
filtration region 305 and reaction detection regions 306 are also
shown.
[0079] Target recognition could be in the form of antibody-antigen
binding, DNA base pair matching, aptamer-target binding, enzymatic
reaction, or any number of other biological reactions. The reaction
could occur on the surface of a substrate, including metal
nanostructures for SERS detection, or on a plastic, metal, or other
material surface. The reaction could occur between gold
nanoparticles for colorimetric, absorption based readouts or
between some combination of a substrate and a nanoparticle for
other detection methods. The modular diagnostic test platform may
contain any form of mechanical, electrical, optical, or other
resonators, or other signal transduction technology, to enhance the
signal of each biomolecular binding reaction. Diagnostic test
platform can be designed to be disposable or washable and
reusable.
[0080] Prototype microfluidic-based, modular diagnostic test
platforms were made of PDMS and glass and plasma-bonded together,
as reported in the literature. Briefly, a mold was made from laser
cut polyacrylic using a Versa laser. The mold was then cast in
PDMS, put under vacuum to removed trapped air bubbles, and baked
overnight. The resulting PDMS cast was cut from the mold. One mm of
PDMS was retained at the end of the sensing channel, enabling a
thin interface for optical measurements along the width of the
platform. Holes were punched at the inlet and outlet and the PDMS
channels and a microscope slide were then plasma treated in an
Oxford Plasma Oxidizer. The treated surfaces were pressed together,
and allowed an hour to bond. Fluidic actuation was then provided
using a syringe and either annual actuation or a Harvard Apparatus
syringe pump.
[0081] An alternative form of a modular diagnostic test platform
704 is schematically illustrated in FIG. 7 (a, b). In this aspect,
the sample collection region 711, a biomolecular reaction region
713, and a control reaction region 715 are disposed serially on the
platform substrate. A sample filtration component 717 is also
illustrated. FIG. 7b further illustrates the stages two and three
shown in FIG. 7a.
[0082] Exemplary targets that have been measured by the inventors
include vitamin D (in the blood) and DNA from Kaposi's sarcoma
associated herpesvirus.
Example I
Quantitative Measurement of 25-Hydroxyvitamin D
[0083] In this example, a modular diagnostic test platform ("test
strip") 704 as shown in FIG. 7 was used. A blood filtration
component 717 was incorporated onto the test strip 704 to filter a
raw blood sample into a serum sample. The filter component 717
comprised a set of stacked commercial filters (Whatman, Kent, UK)
of decreasing pore size filters ranging from approximately 2.7
.mu.m (considered to be about the limit to filter highly deformable
red blood cells) to 0.7 .mu.m (small enough to filter platelets).
(Note that the filter sizes were not optimized for reproducibility,
accuracy, and speed in this work).
[0084] The circulating concentration of 25-hydroxyvitamin D
[25(OH)D], a metabolic product of vitamin D, is considered to be
the best indicator of vitamin D status. We created a nanoparticle
based assay and test strip capable of quantifying 25(OH)D
concentration between 5 ng/mL to 75 ng/mL, spanning both the
healthy and deficient range. The consumable test strip has a
reaction detection region that changes color depending on how much
25(OH)D is present, and a control detection region indicating that
the test has run. FIG. 6 shows the test strip, including both the
detection and control regions and the upstream conjugation pad with
nanoparticles. As shown in detail in FIG. 7, the assay is based
around competitive antibody recognition, similar to non-portable
vitamin D assay. Instead of using an enzymatic or radioactive
label, however, the antibodies are conjugated to 30 nm gold
nanoparticles (Nanopartz, Loveland, Colo.). The nanoparticles have
a strong red color characteristic of their surface plasmon
resonance and high molar absorptivity (3.25 M.sup.-1cm.sup.-1). As
further shown in FIG. 7 following insertion of the test strip in
the accessory, the sample flows downstream (via capillary action)
and mixes with 5 .mu.L of 10 nM nanoparticles stored on the
surface. Some of the nanoparticle conjugates bind to the 25(OH)D
from the sample. The mixture is then transported further downstream
over the detection strip, which contains approximately 20 ug/ml of
immobilized 25(OH)D. For samples with high vitamin D (e.g., >75
ng/mL), most of the nanoparticle antibodies will end up conjugated
to the sample, resulting in only a subtle change in the color of
the detection band. For samples with vitamin D between 75 ng/mL and
5 ng/mL, the band will turn redder, allowing for a quantifiable
readout of vitamin D levels. Samples with vitamin D under 5 ng/mL
saturate the detection band, indicating a severe deficiency.
Downstream past the detection region, a control region is
functionalized with anti-25(OH)D antibodies. These will bind to
passing antibodies indicating the test ran and act as a positive
control.
[0085] Detection regions of the test strip were prepared by coating
a pre-defined quantity of 25(OH)D molecules onto the surface. The
passive adsorption used for most antibody immobilization to surface
could not be used here to yield stable coatings due the small size
of 25(OH)D molecules. Therefore, we used a well-established maleic
anhydride to amine chemistry to covalently immobilize 25(OH)D
molecules. To do so, coating concentrations of 25(OH) D were
enriched with amine functional groups that acted as linkers to the
maleic anhydride activated surfaces.
[0086] Preliminary vitamin D tests to confirm the functionality of
the detection surface and the gold nanoparticle based competitive
detection scheme were carried out using a custom-built chamber with
wells (not shown). Each well, representing a detection zone, had a
7 mm diameter and corresponded to the well size generally used in
commercial 96-well plates. Here, three sets of 10 nM nanoparticle
antibodies were mixed separately with samples of high (75 ng/ml),
low (5 ng/ml) and zero 25(OH)D concentrations. The mixtures were
introduced to the different wells and incubated for four hours.
Following washing, silver enhancer solution (Simga Aldrich) was
introduced and the color development after five minutes was
observed. The intensity of developed color is proportional to the
amount of nanoparticle-antibody conjugates that are bound on the
surface. The nanoparticle antibodies that have interacted with the
higher concentration of 25(OH)D will have the less antibody
conjugates available for binding to the detection surface, which
leads to a lower intensity signal in our test.
[0087] Procedure for obtaining detection surface
[0088] Glass substrate were immersed in 20 mM APTES in isopropanol
for two hours and subsequently annealed at 120.degree. C. for one
hour. One (1) % PSMA dissolved in toluene was spin-coated at 3500
rpm for 30 s followed by curing at 120.degree. C. for two hours.
The glass substrates were then cooled and immersed in acetone for
10 minutes. The 25(OH)D was coated by incubating 20 ug/ml of
aminopropylated 25(OH)D at 37.degree. C. in a humid chamber for
four hours. The surface was washed and the remaining sites were
coated with blocking buffer.
Example II
Detection of Kaposi's Sarcoma Associated Herpes Virus (KSHV)
DNA
[0089] In this example, a modular diagnostic test platform of the
type illustrated as 203 in FIG. 3, and described herein above, was
used.
[0090] Short DNA sequences used as probes for KHSV DNA were
designed using BLAST Primer Design. Briefly, oligonucleotides
specific to KSHV DNA that code for vCyclin were chosen. Gold
nanoparticles were conjugated with oligonucleotides with 5' alkyl
thiol groups. The nanoparticles had an average diameter of 15 nm, a
compromise between the higher sensitivity of larger particles, and
the easier to work with nature and stability of smaller particles.
100 .mu.L of 100 .mu.M KSHV probes were added per 1 mL of 3 nM GNP
solution, and allowed to react overnight. Concentrated solutions of
sodium phosphate and sodium dodecyl sulphate (SDS) were then added
in order to bring the solution to 10 mM and 0.01% concentrations,
respectively, before another overnight period to reaction. Next,
three additions of sodium chloride were added, resulting in
concentration of 100 mM, 200 mM, and 300 mM, each with 24 hours in
between. This process worked to maximize the number of bound DNAs
per particle, making more stable GNP conjugates. The resulting
solution was spun down and resuspended in 0.01% SDS three times to
remove unbound oligonucleotides. Sodium phosphate and sodium
chloride were then re-added to the final solution, resulting in
final concentrations of 10 mM sodium phosphate and 300 mM sodium
chloride.
[0091] GNP solutions of different concentrations were created in
order to calibrate the device. One OD 15 nm GNPs were diluted to
concentrations of (a) 0.8 OD, (b) 0.6 OD, (c) 0.4 OD, (d) 0.2 OD,
and 0.0 OD. The absorbance of these solutions was measured using a
SpectraMAX photometer and the absorbance spectra are shown in FIG.
9b. These solutions were then inserted into the modular diagnostic
test platform via syringe, and measured in order to determine the
smartphone device read-out at these absorbance levels. By mapping
these results to the photometer output, we created a calibration
curve for the smartphone accessory.
[0092] Next, gold nanoparticle conjugates were mixed with different
concentrations of the target KSHV DNA sequence, ranging from 100
.mu.M to 1 .mu.M. After three hours, the solutions were inserted
into the smartphone accessory. The solutions were then measured by
the smartphone, and the results were saved to a connected Google
Drive for analysis.
[0093] Gold nanoparticles and their color change can be seen in
FIG. 9a after the addition of different amounts of KSHV DNA. For
concentrations over 5 nM, a color change reaction was visible. When
no target or little target is present, the nanoparticle solution is
a bright red. As higher concentrations of target DNA are added, the
nanoparticles aggregate, and turn to a duller purple. These color
changes can be measured by the smartphone accessory by using a
light source tuned to the nanoparticles' absorbance peaks (520 nm)
and a photodetector. Further, through changes to the size, shape,
and material the nanoparticles, as well as their concentration, the
sensitivity of the solutions can be tuned. When the optical density
of these solutions is plotted against the absorbance readings taken
from the smartphone accessory (FIG. 9c), a strong linear
relationship was observed.
[0094] In its present configuration, the smartphone accessory had
the ability to determine changes in nanoparticle concentration on
the order of hundreds of pM. Further, simply by changing the light
source and detector, the accessory could be tuned to measure other
color change reactions, such as popular enzymatic ones.
[0095] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0096] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. The term "connected" is to be construed as
partly or wholly contained within, attached to, or joined together,
even if there is something intervening.
[0097] The recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein.
[0098] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate embodiments of the invention
and does not impose a limitation on the scope of the invention
unless otherwise claimed.
[0099] No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0100] It will be apparent to those skilled in the art that various
modifications and variations can be made to the present invention
without departing from the spirit and scope of the invention. There
is no intention to limit the invention to the specific form or
forms disclosed, but on the contrary, the intention is to cover all
modifications, alternative constructions, and equivalents falling
within the spirit and scope of the invention, as defined in the
appended claims. Thus, it is intended that the present invention
cover the modifications and variations of this invention provided
they come within the scope of the appended claims and their
equivalents.
* * * * *